1. Field of the Invention (Technical Field)
The present invention relates to methods and apparatus for nucleic acid determination, more particularly for measurement of nucleic acids (e.g., DNA and RNA), and their sequences and interactions, and for detection of DNA damage, at thick-film electrodes, based on stripping potentiometry.
2. Background Art
Thick-film technology is known to be useful for mass production of miniaturized electrochemical sensors (Prudenziati, S. "Thick-Film Sensors", Elsevier, Amsterdam (1994); Galan-Vidal, C., Munoz, J., Dominguez, C. and Algeret, S., "Trends", Anal. Chem., Vol. 14, p. 225 (1995); and Craston, D., Jones, D., Williams, D. and El Murr, N., Talanta, Vol. 38, p. 17 (1991)). The versatility of modem screen-printing processes, originally developed from microelectronics applications, has led to the automated and semi-automated fabrication of highly reproducible solid-state electrochemical transducers.
Most of the activity in this area has focused on the development of "one-shot" enzyme electrode strips for decentralized clinical testing of metabolites (Alvarez-lcarza, M. and Bilitewski, U., Anal. Chem., Vol. 65, p. 525A (1993); Green, M. and Hildrich, P., Anal. Proceed., Vol. 28, p. 374 (1991)), or field monitoring of pollutants (Kotte, H., Grundig, B., Vorlop, K. D., Strehilz, B. and Stottmeister, U., Anal. Chem., Vol. 67, p. 65 (1995); Skladal, P., Anal. Chim. Acta., Vol. 269, p. 281 (1992)). In addition to biocatalytic sensors, screen-printed electrodes have been fabricated for on-site stripping measurements of trace metals (Wang, J., Tian, B., Anal. Chem., Vol. 65, p. 1529 (1992); Wang, J., Analyst, Vol. 119, p. 763 (1994); U.S. Pat. No. 5,292,423, entitled "Method and Apparatus for Trace Metal Testing", to Wang), or for electrocatalytic-amperometric detection of biomolecules, e.g., glutathione or ascorbic acid (Hart, J., Wring, S. A., Electroanalysis, Vol. 6, p. 617 (1994)).
There is a need for development of a low-cost miniaturized sensing device for the rapid detection of nucleic acids, their interactions, and more particularly their sequences. Such devices hold an enormous potential for early clinical diagnostics of genetically inherited diseases, on-site detection of food-contaminating organisms and forensic or environmental investigations. It is anticipated that both the speed and cost of such a DNA analytical test would be improved dramatically by miniaturization and microfabrication. Silicon-based (thin-film) microlithographic techniques have been explored by various companies for such development of DNA chips. (See Noble, D., "DNA Sequencing on a Chip", Anal. Chem., Vol. 67, p. 201A (1995)).
Electrochemical techniques are known for being useful for nucleic acid analysis in general (Palacek, E., Bioelectrochem. Bioenerg., Vol. 170, p. 421 (1988)), and for sequence-selective biosensing of DNA in particular (Millan, K. M. and Mikkelsen, S. R., Anal. Chem., Vol. 65, p. 2317 (1993); Hashimento, K., Ito, K. and Ishimori, Y., Anal. Chem., Vol. 66, p. 3830 (1994)). However, such early applications have relied on the use of conventional mercury drop or carbon and gold disk electrodes which are bulky and difficult to automate. Additionally, early operations have required time-consuming steps for completion of the task.
It is also known to detect nucleic acid hybridization by direct electrochemical techniques, including performing electrochemical analyses for detecting specific nucleic acids using carbon working electrodes combined with voltammetric procedures (Hall, J., Moore-Smith, J., Bannister, J., and Higgins, l. J., "An Electrochemical Method for Detection of Nucleic Acid Hybridisation", Biochemistry and Molecular Biology International, Vol. 32, No. 1, p. 21 (1994)). A screen-printed electrode is shown in one instance in connection with the detection of hybridization (i.e. native (ds) versus denatured (ss) indicative of hybridization detection), again used with voltammetry. However, these methods and apparatus for DNA/RNA measurement are limited to use with very high concentrations of nucleic acids (e.g., 300 ng) and do not encompass the use of potentiometric stripping analysis (PSA) or related chronopotentiometry for monitoring nucleic acids at screen-printed electrodes, nor the detection of DNA interactions, sequences or damage.
There has been a need for developing reliable methods for detecting and quantifying the human immunodeficiency virus type 1 (HIV-1). The standard diagnostic test for HIV infection is an ELISA for the HIV antibody (in which viral antigens are adsorbed onto a solid phase). Western blot assays have also been used for this task. (Gallo, R., Montagnier, L., Sci. Am., Vol. 259 (10), p. 41 (1988); Kuby, J., Immunology, Chapter 23, W. Freeman Inc., New York (1991); Nishanian, P., Huskins, K., Stehn, S, Deels, R., and Fahey, J., J. Infect. Dis., Vol. 162, p. 21 (1990).) Alternately, nucleic acid hybridization schees have been proposed for detecting HIV-1 DNA sequences. These include radioisotopic assays, with the HIV probe labeled with the .sup.32 p isotope or nonisotopic hybridization procedures employing calorimetric measurements. (Davis, G., Blumeyer, K., DiMichele, L., Whitfield, K., Chappelle, H., Riggs, N., Ghosh, S., Kao, P., Fhay, E., Kwoh, D., Guatelli, J., Spector, S., Richman, D., Gingeras, T., J. Infect. Dis., Vol. 162, p. 13 (1990); Mulder, J., McKinney, N., Christopherson, C., Sninsky, J., Greenfield, L., Kwok, S., J. Clin. Microbiol., Vol. 32, p. 292 (1994); Livache, T., Fouque, B., Teoule, R., Anal. Biochem. Vol. 217, p. 248 (1994); Rapier, J., Villamazo, Y., Schockeman, G., Ou, C., Brakel, C., Jonegan, J., Maltzman, W., Lee, S., Kirtiker, D., Galita, D., Clin. Chem., Vol. 39, p. 244 (1993).) Both of the hybridization strategies rely on prolonged (2-hour to 3-hour) hybridization times, with the isotopic assay complicated by the short half-life and hazardous nature of the radiolabeled probe. Also, although such solution-phase or bead-phase sandwich hybridization assays are suitable for diagnostic laboratories, there is an urgent need for faster, safer, cheaper, and easier-to-use hybridization sensors, based on the integration of DNA recognition layers and physical transducers, for decentralized screening (e.g., self-testing) of HIV-1 DNA.
There is also a long-term need for a rapid and user-friendly sensor for detecting damage to DNA in cells. Damage to DNA in cells leads to serious disturbance of the cell functions, usually involving minor variations in the DNA structure of conformation, and detection of such damage requires a highly sensitive analytical technique. Known procedures for measuring DNA damage rely on lengthy and insufficiently sensitive chromatographic or electrophoretic separation assays (Cadet, J., Weinfeld, M., Anal. Chem., Vol. 65, p. 675A (1993)). In addition, such techniques cannot follow the dynamics of processes occurring in an exposure of DNA to physical or chemical damaging agents. Previous efforts in this area have used polarography for detecting DNA damage induced by exposure to ultraviolet or y radiation; however, since this strategy relies on mercury drop electrodes, it is not suitable for widespread sensing of DNA radiation damage (Vorlikova,., Palecek, E., Int. J. Rad. Biol., Vol. 62, p. 363 (1974); Numberg, et al., Int. J. Rad. Biol., Vol 42, p. 407 (1982)).